Bacteria over-expressing c-di-AMP and therapeutic methods

ABSTRACT

The present invention includes the discovery of a strain of  Mycobacterium  comprising an expression vector encoding a di-adenylate cyclase enzyme. The  Mycobacterium  is selected from the group consisting of  Mycobacterium tuberculosis, Mycobacterium bovis , or a combination thereof and the preferred strain of  Mycobacterium  is BCG. The preferred expression vector is a mycobacterial expression vector including an hsp60 promoter and a DNA sequence of diadenylate cyclase (disA), or a functional part thereof. The strains of  Mycobacterium  are used in therapeutic applications including tuberculosis and cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. national entry ofInternational Application PCT/US2016/017248, having an internationalfiling date of Feb. 10, 2016, which claims the benefit of U.S.Provisional Application No. 62/114,610, filed Feb. 11, 2015, the contentof each of the aforementioned applications is herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant nos.A136973, A137856, A1097138 from the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION-BY-REFERENCE

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

BACKGROUND OF THE INVENTION

Nucleotides are indispensable components of all living cells, as theymake up DNA and RNA, and serve as important energy sources. Nucleotidesalso have key roles in signaling in eukaryotic, bacterial and archaealcells. In bacteria, signaling nucleotides such as cyclic AMP andguanosine tetra- or pentaphosphate ((p)ppGpp) have been classicallylinked to carbon metabolism and the stringent response, which is causedby nutrient limitation. However, it has become clear that signalingnucleotides contribute to the regulation of multiple different pathways;for example, in addition to its involvement in central carbonmetabolism, cAMP is also involved in the regulation of both biofilmformation and virulence gene expression in many pathogenic bacteria. Oneof the latest signaling nucleotides to be identified is cyclic di-AMP(c-di-AMP), which is the second cyclic dinucleotide shown to be producedby bacteria, after cyclic di-GMP (c-di-GMP). It has been suggested thatc-di-AMP and c-di-GMP regulate very different processes.

c-di-AMP is produced from two molecules of ATP by diadenylyl cyclase(DAC) enzymes and is degraded to pApA by phosphodiesterase (PDE)enzymes. The dinucleotide was initially discovered during a structuralstudy on Thermotoga maritima. DNA integrity scanning protein (DisA),which is a homologue of Bacillus subtilis DisA (formerly known as YacK),a bacterial DNA damage checkpoint protein that can delay sporulation inthe event of DNA damage. The first report of c-di-AMP production bybacterial cells came in 2010, when the dinucleotide was identified as amolecule secreted into the cytosol of host cells by the intracellularbacterial pathogen Listeria monocytogenes. Since then, c-di-AMP has beendetected in cellular extracts from Streptococcus pyogenes, B. subtilis,Chlamydia rachomatis and Staphylococcus aureus, and a DisA-typec-di-AMP-synthesizing enzyme from Mycobacterium tuberculdosis has beencharacterized biochemically.

Although most of the mechanistic details still await molecularcharacterization, the regulation of cellular pathways by c-di-AMPpresumably follows the same general principles as for the othersignaling nucleotides. Environmental changes are sensed either directlyor indirectly by the nucleotide-synthesizing or nucleotide-degradingenzymes, leading to a change in the cellular nucleotide concentration.At high concentrations, c-di-AMP is expected to bind to a specific setof receptor or target proteins and allosterically alter their functionor the function of downstream effector proteins, thus controllingspecific cellular pathways. Although many details of the c-di-AMPsignaling network remain to be discovered, this nucleotide has beenlinked to the regulation of fatty acid synthesis in Mycobacteriumsminegatis, to the growth of S. aureus in low-potassium conditions, tothe sensing of DNA integrity in B. subtilis and to cell wall homeostasisin multiple species.

The M. tuberculosis genome encodes a di-adenylate cyclase enzyme (disA,also called dacA; encoded by gene Rv3586 (also called MT3692) in theH37Rv genome or MT3692 in the CDC1551 genome) that synthesizes c-di-AMPfrom ATP or ADP4. Orthologs of disA exist in all mycobacterial genomeswith the exception of M. leprae. However, the role of c-di-AMP in M.tuberculosis physiology and mechanism of its interaction with the hostimmune system is poorly understood. However, the existing model for M.tuberculosis infection is that extracellular mycobacterial DNA is theonly ligand for CSP activation within macrophages, which leads toincreased autophagy and bacterial clearance in an ESX-1 secretionsystem-dependent manner, excluding any role for bacterial CDNs in CSPactivation.

The mammalian innate immune system is composed of receptors thatcollectively serve as a pathogen sensor to monitor the extracellular,vacuolar, and cytosolic cellular compartments. Recognition of microbeswithin these distinct compartments leads to cellular responses that arecommensurate with the microbial threat. Although both pathogenic andnonpathogenic microbes interact with extracellular and vacuolarcompartments, infectious disease agents often mediate their pathogenesisby directly entering the cytosol or through delivery of virulencefactors into the host cell cytosolic compartment. Thus, the innateimmune system may distinguish between pathogenic and nonpathogenicmicrobes by monitoring the cytosol.

Several distinct pathways of innate immunity are present in the hostcell cytosol. One, termed the cytosolic surveillance pathway (CSP),detects bacterial, viral, and protozoan pathogens, leading to theactivation of interferon regulatory factor 3 (IRF3) and nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB), resulting inthe induction of interferon-β (IFN-β) and co-regulated genes. Someligands that activate this pathway are known, for example, viral andbacterial nucleic acids. However, the ligands and host receptors thatlead to IFN-β production after exposure to nonviral microbes-includingL. monocytogenes, M. tuberculoss, F. tularensis, L. pneumophila, B.abortis, and T. cruzi—remain unknown. The mechanisms and role ofc-di-AMP signaling in Mycobacterium tuberculosis infection must beidentified and treatments that prevent, alleviate, or cure tuberculosismust be developed.

Bacille Calmette Guerin (BCG) is the most widely used vaccination in theworld. BCG is made of a live, weakened strain of Mycobacterium bovis, (acousin of Mycobacterium tuberculosis, the TB bacteria). It was developedin the 1930's and it remains the only vaccination available againsttuberculosis today. Despite its protection against active TB inchildren, BCG has failed to protect adults against TB infection andactive disease development, especially in developing countries where thedisease is endemic. Some of key reasons for failure of BCG is lowimmunogenicity and its inability to induce maturation of DC efficiently.Among various strategies that have been employed so far to improve theprotective potential of BCG involve construction of rBCG, which couldconfer similar or higher protection along with induction of a betterimmunological memory than BCG. Most of the methodologies used to achievegreater immunogenicity involve (i) over-expression of promisingimmuno-dominant antigens either singularly or as fusion with otherimmuno-dominant antigens, (ii) over-expression and reintroduction ofantigens lost during the attenuation process or (iii) over-expression ofmammalian cytokines in BCG such as IL-2, IL-12, IL-15, and GM-CSF. Newmethods of tuberculosis vaccination are needed to prevent the spread ofdisease.

In addition, more than 60,000 new cases of bladder cancer are diagnosedeach year in the United States accounting for approximately 13,000deaths. BCG-based therapy is currently the most effective intravesicaltherapy for nonmuscle invasive bladder cancer (NMIBC) and it representsthe only agent known to reduce the progression of invasive bladdercancer into muscle. It is widely accepted that an intact immune systemis a prerequisite to a successful therapy. BCG-induced antitumor effectsdepend on a sequence of events involving a complex interplay of solubleand cellular immune mediators and a cross-talk between innate andadaptive immunity. Limitations of BCG therapy include recurrence of thedisease after initiation of BCG therapy. Consequently, new BCG strainsenhancing the prevention or cure, and minimizing the recurrence rate, ofcancer in patients must be identified.

SUMMARY OF THE INVENTION

One embodiment of the invention is a pharmaceutical compositioncomprising a compound, salt, solvate, or stereoisomer of a syntheticc-di-AMP, and a pharmaceutically acceptable carrier. This pharmaceuticalcomposition may include at least one or more other compounds enhancingimmunogenicity such as mycobacterial DNA, IFN, or c-di-AMP andcombinations thereof.

Another embodiment of the invention is a method of treating a bacterialinfection in a subject comprising administering to the subject aneffective amount of a compound, salt, solvate, or stereoisomer ofsynthetic c-di-AMP.

Another embodiment of the invention is a method of treating tuberculosis(TB) in a subject comprising administering an effective amount of acompound, salt, solvate, or stereoisomer of c-di-AMP.

Another embodiment of the invention is the discovery of one or morestrain(s) of Mycobacterium comprising an expression vector encoding adi-adenylate cyclase enzyme. The Mycobacterium is preferably selectedfrom the group consisting of Mycobacterium tuberculosis, Mycobacteriumbovis, or a combination thereof. The preferred strain of Mycobacteriumbovis is a Mycobacterium bovis bacille Calmette-Guérin strain (“BCG”)that preferably over-expresses a diadenylate cyclase (disA) of M.tuberculosis (Rv3586) from a mycobacterial expression vector (orplasmid). The most preferred strains are BCG-pSDhsp60.MT3692 (a BCGstrain harboring the episomal plasmid pSDhsp60.MT3692), andBCG-pMH94Hyg.MT3692 (a BCG strain harboring the integrative plasmidpMH94Hyg.MT3692). Many strains of BCG maybe transformed with plasmids ofthe present invention, pSDhsp60.MT3692 and pMH94Hyg.MT3692 to form novelpharmaceutical compositions. The preferred mycobacterial expressionvector includes an hsp60 promoter and a DNA sequence of diadenylatecyclase (disA), or a functional part thereof, wherein the expression ofdi-adenylate cyclase enzyme or a functional part thereof is regulated bythe hsp60 promoter. The term “functional part thereof” means a part ofthe diadenylate cyclase enzyme that maintains its enzymatic activity.The one or more strains of Mycobacterium described above are used intherapeutic applications including tuberculosis and cancer, specificallynonmuscle invasive bladder cancer.

Another embodiment of the invention is a pharmaceutical composition ofthe one or more strain(s) of Mycobacterium described above and apharmaceutically acceptable carrier. This pharmaceutical composition maybe combined with at least one or more compounds enhancing immunogenicitydescribed above.

Another embodiment of the invention is a method of vaccinating a subjectagainst TB comprising administering to the subject and effective amountof the one or more strain(s) of Mycobacterium described above and apharmaceutically acceptable carrier. This method of vaccination may alsoinclude one or more compounds enhancing immunogenicity described above.

Another embodiment of the invention is a method of treating orpreventing cancer in a subject comprising administering to the subjectan effective amount of the one or more strain(s) of Mycobacteriumdescribed above and a pharmaceutically acceptable carrier. This methodof treating or preventing cancer may also include one or more compoundsenhancing immunogenicity described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D illustrates the c-di-AMP production by M. tuberculosis in thein vitro broth culture. Mycobacterium tuberculosis stain CDC1551 wasgrown in Middle Brook 7H9 broth supplemented with 10% OADC, 0.5%glycerol and 0.05% Tween 80 and at various stages of growth, nucleotideswere extracted and analyzed for the presence of c-di-AMP. LC-MS-MRMchromatograms of (a) intracellular (intra-bacterial) and (b) secreted(culture supernatant) c-di-AMP detected from M. tuberculosis culture atan O.D. (A_(600 nm)) of 1. (c) Intra-bacterial levels of c-di-AMPextracted from 2×10⁸ bacilli at the indicated culture O.D. Data is Meanplus/minus SD of triplicate samples. (d) intra-macrophage (per 6×10⁶J774 cells) levels of M. tuberculosis secreted c-di-AMP at 24 hr afterinfection with an MOI of 1:20. Data is Mean plus/minus SD of triplicatesamples.

FIG. 2A-2D illustrates generation and confirmation of M. tuberculosisover expression. (a) The disA gene of M. tuberculosis, MT3692, wasPCR-amplified from M. tuberculosis CDC1551 genomic DNA usinggene-specific primers. The amplicons were cloned into the mycobacterialexpression Vector pSD5-hsp60 at the Nde I and Mlu I restriction sites.(b) The resulting construct pSD5-hsp60-MT3692 was sequenced and used totransform M. tuberculosis CDC1551 and recombinant clones were selectedagainst Kanamycin (25 ug/ml). Colony PCR using Kanamycin gene specificprimers confirmed the presence of recombinant plasmid in the Mtb-OEstrain. (c) Table depicting the RNAseq. result of Mtb-CDC151 and theMtb-OE stains for the gene MT3692 indicating approximately 95 foldover-expression of MT3692 in Mtb-OE in comparison to Mtb-CDC1551. ForRNA sequencing, total RNA were extracted from M. tuberculosis culturesat an O.D. of 1, followed by rRNA depletion and sequencing using AppliedBiosystems SOLID™ Titak RNA-Seq Kit protocol on a AB 5500xl SOLIDsequencer. (d) Comparison of the intra-bacterial levels of c-di-AMPextracted from 2×10⁸ cells of Mtb-CDC1551 and Mtb-OE at culture O.D.of 1. Data is Mean plus/minus SD of triplicate samples; ***, p<0.001 byStudent's t-test (2-tailed).

FIG. 3A-3C illustrates transposon insertion mutant of Mycobacteriumtuberculosis CDC 1551 for MT3692 gene is unable to produce c-di-AMP.Mycobacterium tuberculosis strain CDC1551 and Mtb-disA-KO (JHU-3586)were grown in Middle Brook 7H9 broth supplemented with 10% OADC, 0.5%glycerol and 0.05% Tween 80 and nucleotides were extracted frombacterial pellet at culture O.D. of 1. LC-MS-MRM chromatograms ofintracellular c-di-AMP extracted from (a) Mtb-CDC1551 and (b)Mtb-disA-KO. (c) Comparison of the intra-bacterial levels of c-di-AMPextracted from 2×10⁸ cells of Mtb-CDC1551 and Mtb-disA-KO at cultureO.D. of 1 by LC-MS-MRM confirms that Mtb-disA-KO stain is devoid ofc-di-AMP production.

FIG. 4A-4D illustrates generation and confirmation of M.tuberculosis-CDC 1551 MT3692 complemented strain (Mtb-COMP), (a)Organization of radA-disA operon in genome of M. tuberculosis CDC1551.(b) The whole operon of rad4-disA including the upstream promoter regionwas PCR-amplified using M. tuberculosis CDC1551 genomic DNA and primersOPE-MT3692(F) and OPE-MT3692(R) and cloned into an integrative vector,pMH941-Hyg, at XbaI-restriction site. (c) The resulting construct,pMH94Hyg-MT3692 was used to transform the Mtb-disA-KO strain (JHU-3586).Candidate Hyg^(R) colonies were selected and genomic DNA from theMtb-COMP clones were used for PCR based confirmation using Hygromycingene specific primers. (d) Comparison of the intra-bacterial levels ofc-di-AMP extracted from 2×10⁸ cells of Mtb-disA-KO and Mtb-COMP atculture O.D. of 1 by LC-MS-MRM confirms the reconstitution of c-di-AMPproduction in the Mtb-COMP strain.

FIG. 5 illustrates Interferon Regulatory Factor (IRF) pathway activationfollowing infection with various M. tuberculosis strains with variedlevels of c-di-AMP production. THP1-Dual™ cells (InvivoGen) were grownas per the supplier's recommeridations. Twenty four hours afterinfection of PMA activated THP-Dual™ cells with a pre-calibrated MOI of1:10, culture supernatants were separated and used for measurement ofIRF pathway activation by Quanti-Luo™ assay. Data are mean plus/minus SEat the least three experiments (n=3); *, p<0.05, ***, p<0.001 byStudent's test (2-tailed).

FIG. 6A-6F illustrates increased induction of pro-inflammatory cytokinesfollowing infection with the c-di-AMP over-expressing M. tuberculosisstrain. Levels of (a,b) IL-1α, (c,d) IL-6 and (e,f) TNF-α in culturemedia at 24 h post-infection from BMDM and BMDC cells infected withvarious M. tuberculosis strains at an MOI of 1:10. Data are meanplus/minus SE of at the least three experiments (n=3). *, p<0.05 and **,p<0.01 by Student's t-test (2-tailed).

FIG. 7 illustrates in vitro growth pattern of M. tuberculosis strainspossessing different c-di-AMP production levels. Mycobacteriumtuberculosis strains were grown in Middle Brook 7H9 broth supplementedwith 10% OADC, 0.5% glycerol and 0.05% Tween 80 for 20 days. At theindicated time points after inoculation, samples were collected andabsorbance (A₆₀₀) were measured by spectrophotometer. Comparable growthwas observed amongst all the M. tuberculosis strains. Data are mean oftriplicate samples. Error bars are overlapping and not shown for clarityof the line diagram.

FIG. 8A-8B illustrates increased phosphorylation of TBK1 in macrophagefollowing infection with the Mtb-OE strain. (a) Fluorescence microcopymerged (Blue+Green channel) images of J774 cells, fixed after 6 hr ofinfection with various M. tuberculosis strains and stained withanti-pTBK1 antibody; Nuclei-Blue (DAPI), pTBK1-Green (AF488). (b)Quantitative analysis of pTBK1 positive staining of the infectedmacrophage cells revealed significantly higher percentage of cellexhibiting increased staining for pTBK1 in case of Mtb-OE infection incomparison to infection with other Mtb-strains. Box plot depicting Mean(+), Median (−) with quartiles (box margins) and ranges (bars) (n=6). *,p<0.5 and ***, p<0.001 by Student's t-test (2-tailed).

FIG. 9A-9D illustrates knock down of DDX41 in macrophage leads toreduced induction of type I IFN and TNF-α response. (a) Western blotanalysis of DDX41 and GADPH (loading control) of DDX41 Knock down (KD)and control (WT) RAW-Bue™ ISG cells and densitometry analysis of thewestern blots showing approximately 30% reduced DDX41 protein level inthe KD cells. Data are mean from duplicate experiments. (b) As anindicator of IRF induction, levels of SEAP in the supernatant of DDX41Knock down (KD) and control (WT) RAW-Blue™ ISG cells at 18 hr afterinfection with various M. tuberculosis strains were measured by aspectrophotometer following Quanti-Blue™ assay. (c) Levels of IFN-β and(d) TNF-α in culture media of DDX41 Knock down (KD) and control (WT)RAW-Blue™ ISG cells at 18 hr after infection with various M.tuberculosis strains at MOI of 1:5 were measured by ELISA. Data are meanplus/minus SE of at the least three experiments (n=3). **, p<0.0.5 and***, p<0.001 by Student's t-test (2-tailed).

FIG. 10A-10H illustrates modulation of host cytokine response andintracellular growth of M. tuberculosis by c-di-AMP. (a) Levels of IFN-βin culture media at 24 h post-infection from resting and (b) IFN-β/LPSactivated J774.1 cells infected with various Mtb-strains at an MOI of1:20. (c) Levels of TNF-α in culture media at indicated time points fromresting and (d) IFN-β/LPS activated J774.1 cells infected with variousMtb-strains at an MOI of 1:20. (e) Levels of IFN-β in culture media at24 h post-infection from BMDM and (f) BMDC cells infected with variousMtb-strains at an MOI of 1:10. ELISA Data are mean±SE of at the leastthree experiments (n=3). *, p<0.05; **, p<0.01 and ***, p<0.001 byOne-way ANOVA with Tukey's post test. (g) IFN-β mRNA were assessed byqRT-PCR in BMDCs infected with various Mtb-strains at 24 hpost-infection; data are mean±SD (n=3) and representative of twoexperiments. **, p<0.01 and ***, p<0.001 by One-way ANOVA with Tukey'spost test. (h) Growth kinetics of various Mtb-strains in resting and (i)IFN-□/LPS activated J774.1 cells. Data are Mean CFUs±SD at each timepoint (n=3) and representative of two experiments. Bar diagrams (rightpanels in h and i) represent Mean CFUs±SD at Day 4. *, p<0.05 and ***;p<0.001 by Student's t-test (2-tailed).

FIG. 11A-11C illustrates c-di-AMP produced by M. tuberculosis inducesautophagy in macrophage cells. (a) Fluorescence confocal images ofJ774.1 cells, fixed after 6 hr of infection with various M. tuberculosisstrains and stained with anti-LC3 antibody; Nuclei-Blue (DAPI),LC3b-Green (AF488). Scale bars depicts 20 μm for 40× images and 10 μmfor 100× images. (b) Quantitative analysis of LC3 positive J774.1 cellsshowing puncta formation. Only those cells were considered as positiveand included for quantification, which exhibited formation of large LC3aggregates occupying area >1 μm, Percentage of LC3-II positive cellswere calculated and data are depicted by box plot indicating Mean (+),Median (−) with quartiles (box margins) and ranges (bars) (n=9). *,p<0.05; **, p<0.01 and ***, p<0.001 by One-way ANOVA with Tukey's posttest. (c) Western blot analysis of LC3-I and LC3-II and GAPDH (loadingcontrol) of J774.1 cells at 6 hr after infection along with bar diagramdepicting densitometric ratios of normalized LC3-II/LC3-1 levels, Dataare mean±SD (n=2) from two experiments.*, p<0.05 by Student's t-test(2-tailed).

FIG. 12A-12H illustrates attenuation of virulence and pathogenicity inc-di-AMP over-producing M. tuberculosis strain. (a) Survival of mice(n=10) following infection with various Mtb-strains. ***, p<0.001 byLog-rank (Mantel-Cox) test. (b) Growth kinetics of various M.tuberculosis-strains in mouse lungs and (c) spleen after aerosolinfection. Data are mean±SE (n=4). *, p<0.05; **, p<0.01 and ***,p<0.001 by Two-way ANOVA with Bonferroni post-test. (d) Gross and (e)histo-pathological features of lungs and spleen of mouse infected withvarious M. tuberculosis-strains. Scale bar is 100 μm. (t) Levels ofIFN-β, (g) TNP-α and (h) IFN-β in the serum of mice infected with M.tuberculosis-strains possessing varied ability to produce c-di-AMP. Dataare mean±SE (n=4). *, p<0.05; **, p<0.01 and ***, p<0.001 by Student'st-test (2-tailed).

FIG. 13A-13H illustrates contribution of STING and cytosolic DNAreceptor cGAS to c-di-AMP mediated activation of IFN-pi during M.tuberculosis infection. (a, c) IRF pathway activation as measured byluciferase reporter assay and (b, d) IFN-3 levels in the 18 hpost-infection (MOI=1:5) and post-stimulation culture supernatants ofmouse RAW264.7 derived STING ablated [STING-KO] and control [WT]macrophage IRF reporter cells. (e) IFN-β induction in BMDMs and (f)BMDCs from control [WT] and cGAS ablated [cGAS-KO] mouse followinginfection (MOI=1:10) with various Mycobacterium strains. (g) c-di-AMPconcentration dependent induction of IFN-β in mouse BMDMs. Data aremean+SE of at the least three experiments (n=4 in a, b; n=3 in c, d, e,f g).*, p<0.05; **, p<0.01 and ***, p<0.001 by Student's t-test(2-tailed). (h) Levels of IFN-β mRNA were determined by real-time RT-PCRIn BMDCs derived from wild type cGAS sufficient [WT] and cGAS ablated[eGAS-KO] mouse following infection (MOI=1:10) with variousMycobacterium strains. The IFN-β mRNA expression levels were normalizedto -actin expression and are represented relative to those of untreatedcells. Data are mean±SD (n=3) and is representative of two experiments.*, p<0.05; **, p<0.01 and ***, p<0.001 by Students t-test (2-tailed).

FIG. 14 illustrates plasmids and Mycobacterium used in the invention.

FIG. 15A-15B illustrates a) Mice experiments with a strain of BGCincluding an expression vector encoding a diadenylate cyclase protein(rBCG-disA) and b) Graph of CFU one day post infection.

FIG. 16 illustrates the gross pathology of organs 18 weeks postinfection from mice described in FIG. 15.

FIG. 17A-17B illustrates a) lung and b) spleen CFU 18 weeks postinfection from mice described in FIG. 15.

FIG. 18A-18B illustrates the a) prophylactic potential of BCG strainincluding a DNA expression vector encoding disA (rBCA-disA) in guineapigs and b) CFU one day post infection.

FIG. 19 illustrates the gross pathology and CFU of lungs 14 weekspost-infection of the guinea pigs described in FIG. 18.

FIG. 20 illustrates the gross pathology and CFU of spleen 14 weekspost-infection of the guinea pigs described in FIG. 18.

FIG. 21 illustrates the gross pathology of lungs 18 weeks post-infectionof the guinea pigs described in FIG. 18.

FIG. 22 illustrates the gross pathology of spleen 18 weekspost-infection of the guinea pigs described in FIG. 18.

FIG. 23 illustrates the graphs of lung and spleen infection of theguinea pigs described in FIG. 18.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIG. 1, M. tuberculosis produced and secreted c-di-AMP. Theamount of c-di-AMP was quantified in a 7H9 broth culture and within thebacteria (intracellular). Intracellular c-di-AMP levels were observed toincrease during late-log and stationary phases of growth of M.tuberculosis compared to early log phase growth. After 24 hours ofinfection of J774 mouse macrophage cells with M. tuberculosis, thec-di-AMP produced by the bacteria was detected in the macrophage cytosolof the J774 cells.

Strains of M. tuberculosis, producing different amounts of c-di-AMP,were formed and then used in studies of the present invention. Asillustrated in FIG. 2, a recombinant M. tuberculosis strain Mtb-OE (OEmeans “over expression” of c-di-AMP) was formed having over 95-foldexpression of an endogenous di-adenylate cyclase gene, disA, and aresultant increase in the production of c-di-AMP by ˜20 fold whencompared to the M. tuberculosis CDC1551 a wild type (WT) strain of M.tuberculosis. As described in SUPP FIG. 3, a recombinant M. tuberculosisstrain Mtb-disA-KO (KO means “Knock Out” of the disA gene) was producedwith a transposon insertion disrupting the di-adenylate cyclase domainof disA making the strain substantially free of c-di-AMP. c-di-AMP isproduced by a single di-adenylate cyclase in M. tuberculosis so knockingout this gene knocks further knocks out c-di-AMP production. As shown inFIG. 4, a strain Mtb-COMP was formed by taking the Mtb-disA-KO strainand transforming it with an expression vector with an endogenous disAgene and native promoter. The addition of the expression vectorreconstituted c-di-AMP production in Mtb-disA-KO.

As shown in FIG. 10, J774.1 mouse macrophage cells were infected withthese M. tuberculosis strains (Mtb-CDC1551, Mtb-disA-KO, Mtb-COMP, andMtb-OE) expressing different amounts of c-di-AMP in vitro. Afterdifferent incubation times, the amount of IFN-β produced by the J774.1cells were measured. As shown in FIG. 10, infection with the Mtb-disA-KOstrain resulted in a significant reduction in IFN-3 induction by J774.1cells compared to infection with the Mtb-CDC1551. Conversely, infectionwith the Mtb-OE strain resulted in an enhanced induction of IFN-β byboth resting and activated 1774.1 cells. Notably, Mtb-OE infected cellsalso secreted significantly higher levels of TNF-α compared to theMtb-WT infected cells (or Mtb-CDC1551), whereas Mtb-disA-KO infectedcells produced lower TNF-α levels compared to other groups (FIG. 10c, d). As shown in SUPP FIG. 5, the patterns of Interferon Regulatory Factor(IRF) pathway activation in THP1-human monocyte cells was analyzed andIFN-β responses in mouse primary bone marrow derived macrophages (BMDM)and dendritic cells (BMDC) (FIG. 10e, f ) were comparable. However, themouse BMDCs are a comparatively better IFN-β producer than BMDMs inresponse to M. tuberculosis infection. Induction of IFN-β was furtherconfirmed by real time RT-PCR of the BMDC cells infected with thevarious M. tuberculosis strains (FIG. 10g ). We also observed inductionof significantly higher levels of pro-inflammatory cytokines includingIL-1α, IL-6 and TNP-α by both BMDMs and BMDCs following infection withthe c-di-AMP over-expressing M. tuberculosis strain Mtb-OE (FIG. 6).These observations suggest that perturbation of c-di-AMP levels in M.tuberculosis not only influences the CSP mediated Type I IFN responsebut also plays a critical role in modulating the pro-inflammatorycytokine signature of the infected cells.

Taking into account the ambiguous role of the Type I IFN response inhost control of TB, the growth patterns of these M. tuberculosis strainsin resting and IFN-β/LPS activated J774.1 cells were monitored. Whileall M. tuberculosis WT and recombinant strains exhibited identicalgrowth rates in 7H9 broth culture (FIG. 7), the Mtb-OE strain overexpressing c-di-AMP exhibited significantly diminished intracellulargrowth compared with the other M. tuberculosis strains (FIG. 10h, i ).Growth attenuation of the knock out strain Mtb-disA-KO which is c-di-AMPdeficient strain was not noticed. These observations reveal thatover-expression of c-di-AMP by M. tuberculosis results in significantattenuation of the intracellular growth of the Mtb-OE strain.

Next, we investigated whether enhanced macrophage autophagy mightaccount for the attenuation of the c-di-AMP over-expressing M.tuberculosis strain Mtb-OE by examining the auto-phagosome membranespecific marker LC3 in M. tuberculosis infected J774.1 cells.Fluorescence confocal imaging demonstrated a considerably higherpercentage of cells (˜15%) exhibiting LC3 puncta formation in the caseof the over expression c-di-AMP strain Mtb-OE infection compared to thewild type strain Mtb-CDC1551 (˜10%) and the knock out strain Mtb-disA-KOwhich is c-di-AMP deficient (˜6%) (FIG. 11 a, b). In addition, Westernblot analysis of endogenous LC3 revealed an increase in conversion ofLC3-I to LC3-II in the Mtb-OE infected cells, indicatinghyper-activation of autophagy (FIG. 2c ). We also observed aconsiderably higher percentage of cells exhibiting pTBK1 positivitysuggesting activation of IRF pathway in the Mtb-OE infected 1774.1 cells(FIG. 8). These observations strongly suggest that hyper-induction ofautophagy by macrophages may be one of the contributing factors thatrestricts the intracellular growth of Mtb-OE strain.

The virulence and pathogenicity of the M. tuberculosis strains in mouseaerosol infection models were examined. Remarkably, compared to WTinfection using strain Mtb-CDC1551 (median time to death [MTD] of 150days), a significant increase in the survival of Mtb-OE infected mice(MTD 321 days) was observed. In contrast, the knock out strainMtb-disA-KO which is c-di-AMP deficient showed reduced survival with anMTD of 77 days (FIG. 12a ). Concomitantly, the Mtb-OE strain alsoexhibited growth attenuation as evidenced by significantly reduced lungand spleen bacillary loads (FIG. 12b, c ). Gross and histo-pathologicalfindings of mouse lungs and spleens correlated well with the bacterialorgan burden observations (FIG. 12d, e ). The lungs of Mtb-OEinfected-mice showed significantly fewer and smaller tubercle-likelesions compared to other groups. Concordantly, while Mtb-CDC1551,Mtb-disA-KO, and Mtb-COMP strain-infected mice exhibited considerablesplenomegaly, spleens of the Mtb-OE infected mice appeared normal insize (FIG. 12d ). Altogether, these observations clearly demonstrateattenuation of virulence in the c-di-AMP over-expressing M. tuberculosisstrain.

The mouse serum cytokine levels between these groups at an early stageof disease of 2 weeks post-infection were compared. Consistent with thein vitro studies in mouse and human cells, we observed increased IFN-βlevels in the serum of Mtb-OE infected mice compared to the Mtb-CDC1551and Mtb-disA-KO group (FIG. 12f ). In addition, the Mtb-OE groupexhibited significantly higher serum levels of TNF-α (FIG. 12g ). SinceType I IFN (IFN-β) is known to counter-regulate Type II IFN (IFN-β)responses, we measured IFN-β levels in the serum of infected mice (FIG.12h ). A strong inverse relationship between IFN-β and IFN-γ in thesemice corresponds to the ability of the M. tuberculosis-strains toproduce c-di-AMP.

These studies revealed bacterial c-di-AMP levels are strongly associatedwith the immunopathological outcome of bacterial infection, including M.tuberculosis, in mice. Next, the host cytosolic sensors that may detectM. tuberculosis-derived c-di-AMP starting with helicase DDX41, acytosolic DNA, and CDN receptor that signals via STING were examined. AshRNA-mediated knocked down of DDX41 using RAW-Blue™ ISG cells(InvivoGen) that allow colorimetric measurement of the induction of theIRF pathway was performed. Knockdown of DDX41 caused a significantdefect in activation of the IRF pathway and reduced IFN-β inductionfollowing infection with all the M. tuberculosis strains (FIG. 9 a, b,c). We also observed a significantly reduced TNF-α production by theDDX41 knock-down cells (FIG. 9d ). These results suggest that DDX41 is akey pattern recognition receptor for both DNA and c-di-AMP, and thatDDX41 regulates the induction of Type I IFNs as well as TNF-α followingM. tuberculosis infection.

The contribution of STING to the c-di-AMP-mediated IFN-β response duringM. tuberculosis infection was investigated. A partial knock-down ofSTING in human THP1 cells showed considerably lower IFN-β induction thancontrol cells (FIG. 10). Moreover, all M. tuberculosis strains failed toactivate the IRF pathway or induce IFN-β in mouse RAW 264.7 macrophageIRF reporter cells lacking STING (STING-KO) (FIG. 13 a, b, c, d).However, LPS, which stimulates Type I IFN through STING-independentpathways, induced elevated IFN-β response even in STING-KO cells (FIG.13a, b ). These results confirm that, in addition to its role inbacterial DNA mediated responses, STING is an essential component forc-di-AMP-mediated activation of the IRF pathway during M. tuberculosisinfection. Furthermore, infection of macrophages with M. bovis bacilleCalmette-Guérin (BCG), which is known to lack the Esx-1 secretionsystem, also showed activation of the IRF pathway at levels 20-60% ofthose seen following infection with either the Mtb-CDC1551 or the Erdmanstrain, a WT M. tuberculosis strain considered to be highly virulent(FIG. 13a, b ). Importantly, the c-di-AMP over-expressing M, bovis BCGstrain (BCG-OE) produced a significantly higher IRF and IFN-β responsethan M. bovis BCG itself thus strongly suggesting that bacterial-derivedc-di-AMP gains access to the host cell cytosol despite the absence of anEsx-1 secretion system (FIG. 13a, b ). These experiments indicate that,while contributory to overall type I IFN response, Esx-1 may not beessential for c-di-AMP-triggered IRF pathway activation. However,further studies with ESX-1 deleted M. tuberculosis strains may providedirect evidence for contribution of ESX-1 secretion system in c-di-AMPmediated responses during M. tuberculosis infection.

Next, the role of cGAS in the detection of bacterial c-di-AMP wasexamined. Primary BMDMs (FIG. 13e ) and BMDCs (FIG. 4f ) from WT andcGAS-KO mice22 were infected with these mycobacterial strains and thenIFN-β protein levels were measured. While loss of cGAS resulted in aconsiderably reduced IFN-03 response compared to cells with intact cGAS(WT), all c-di-AMP overproducing strains continued to show significantlyhigher induction of IFN-β in cGAS-KO cells compared to their respectiveWT mycobacterial strains (FIG. 13e, f ). Further, both WT and cGAS-KOBMDMs produced comparable levels of IFN-β following stimulation withsynthetic c-di-AMP (FIG. 13g ). Real time RT-PCR for IFN-β in BMDCsfurther confirmed these results (FIG. 13h ). These experiments show thatwhile c-di-AMP is a key ligand for IFN-β induction irrespective of cGAS,a significant part of the overall IFN-β response during M. tuberculosisinfection is cGAS dependent and hence is probably due to bacterial DNA.

The data thus revealed the involvement of c-di-AMP as an M. tuberculosisPathogen Associated Molecular Pathway (PAMP) that triggers host cellIFN-β secretion and autophagy. Our findings, which employed multiplebacterial strains (including the wild type M. tuberculosis CDC1551 andErdman strains, and M. bovis BCG) were each modified to overexpressc-di-AMP and a variety of host phagocytic cells including thosedefective in important mediators of the CSP (STING, DDX41, and cGAS),consistently demonstrated that c-di-AMP, not bacterial DNA alone, is akey mediator of Type I IFN responses. Supplementary Table 1 lists majordifferences in our methods compared with those of earlier studies andreveals that strain, host cell, and methodological differences may haveallowed the importance of c-di-AMP to have been overlooked in earlierstudies. The studies have shown that c-di-AMP enhances the induction ofType I IFN in subjects as well as several pro-inflammatory cytokinesincluding IL1-α, TNF-α and IL-6 that are believed to play protectiveroles during bacterial infections such as a M. tuberculosis infection.The data illustrates that resistance to tuberculosis (TB) requiresCSP-mediated detection of c-di-AMP produced by M. tuberculosis and thatlevels of c-di-AMP modulate the fate of infection. A di-adenylatecyclase (disA or dacA)⁴ over-expressing M. tuberculosis strain wasformed that secretes excess c-di-AMP and activates the interferonregulatory factor (IRF) pathway with enhanced levels of IFN-β, elicitsincreased macrophage autophagy, and exhibits significant attenuation inmice. c-di-AMP-mediated IFN-β induction during M. tuberculosis infectionwas shown to require stimulator of interferon genes (STING)⁵-signaling.c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acidreceptor cyclic-GMP-AMP (cGAMP) synthase (cGAS), but cGAS neverthelesscontributes substantially to the overall IFN-β response to M.tuberculosis infection. The present invention demonstrates c-di-AMP tobe a key mycobacterial pathogen associated molecular pattern (PAMP)driving host Type I IFN responses and autophagy. Modulating the levelsof c-di-AMP in a subject will enhance the subject's immune response andmay be used to treat disease including immune-deficient disease such asHIV and bacterial infections including TB.

Hence, in this study we generated a recombinant BCG that over-expressesdiadenylate cyclase (disA) of M. tuberculosis (Rv3586) and tested theprophylactic potential of rBCG-disA as a vaccine in mouse and guinea pigmodel of aerosol M. tuberculosis infection and also tested its abilityto induce Type I IFN response. BCG strains modified to over-expressc-di-AMP exhibited marked improvement in protective immunity againsttuberculosis as evidenced by marked reduction in lung and spleenbacillary load and reduced pathology in guinea pig and mouse models ofinfection. In addition, in vitro studies in RAW cells revealed that, ac-di-AMP-over-expressing BCG strain (rBCG-disA) produced a significantlyhigher IRF activation and IFN-β response than BCG itself, suggestingthat bacteria-derived c-di-AMP gains access to the host cell cytosoldespite the absence of an ESX-1 protein secretion system in the BCGstrain and can potentiate the ability of BCG to induce higher IFN-βresponse.

Methods

In this study we generated a recombinant BCG that over-expressesdiadenylate cyclase (disA) of M. tuberculosis (Rv3586) and tested theprophylactic potential of rBCG-disA as a vaccine in mouse and guinea pigmodel of aerosol M. tuberculosis infection and also tested its abilityto induce Type I IFN response and dependence on STING (Stimulator ofInterferon Genes) and cGAS (cyclic GAMP Synthase) signaling axis.

Results BCG strains modified to over-express c-di-AMP exhibited markedimprovement in protective immunity against tuberculosis as evidenced bymarked reduction in lung and spleen bacillary load and reducedpathology. In addition, in vitro studies in RAW cells revealed that, ac-di-AMP-over-expressing BCG strain (rBCG-disA) produced a significantlyhigher IRF activation and IFN-β response than BCG itself in a STINGdependent and cGAS independent manner, suggesting that bacteria-derivedc-di-AMP gains access to the host cell cytosol despite the absence of anESX-1 protein secretion system in the BCG strain and can potentiate theability of BCG to induce higher IFN-β response.

We hypothesized that over-production of c-di-AMP by BCG may offer amulti-pronged approach to tap the adjuvant potential of c-di-AMP toimprove the protective potential of BCG via (i) enhancing the type I IFNand other pro-inflammatory cytokine responses compared to BCG; (ii)enhancing the intrinsic ability of BCG to cause DC maturation; (iii)enhancing over-all antigen presentation following BCG vaccination viainduction of higher levels of autophagy and induction of co-stimulatorymolecules by this rBCG. The method disclosed in the present inventiondepends on the over-production of c-di-AMP by rBCG-disAOE. The presentinvention thus, provides a novel way to improve the existing BCG vaccineintrinsically without the need of exogenous addition of cytokines or useof synthetic chemicals or nucleotide molecules.

The present invention provides an improved method of immunizationagainst tuberculosis using recombinant BCG-disAOE (rBCG-disAOE). Themethod disclosed in the present invention depends on the over-productionof c-di-AMP by rBCG-disAOE. Mycobacterium bovis BCG Pasteur strain overexpressing disA (Rv3586) gene of Mycobacterium tuberculosis under thetranscriptional control of a strong mycobacterial promoter hsp60 usingmycobacterial vectors described (DasGupta, Jain et al. 1998; Dhar, Raoet al. 2000; Jain, Dey et al. 2008). In the present invention, theprotective efficacy of rBCG was assessed in mouse and a highlysusceptible guinea pig model against M. tuberculosis challenge by theaerosol route as described before (Jain, Dey et al. 2008; Dey, Jain etal. 2011).

Immunization of mouse with rBCG resulted in a significantly enhancedprotection characterized by a marked reduction in bacillary load inlungs (1.162 logic) and spleen (0.72 logic) compared to sham-immunizedmice at 10 Weeks post-infection. However, at this time point differencesin CFU were not significantly different from BCG. Further at 18 weekspos-infection, the extent of reduction in lung CFU in case ofrBCG-disAOE immunization was markedly greater when compared to sham (by0.35 log₁₀) and BCG (by 0.49 log₁₀), immunized animals signifyinggreater protection against pulmonary disease.

Most significant effect of rBCG immunization on disease control, both interms of reduction in bacillary load and pathology was evident in guineapig model of M. tuberculosis infection. Wherein, immunization with bothBCG as well as with rBCG-disAOE resulted in a significant reduction inlung and spleen bacillary load, when compared to the saline treatedanimals at 14 weeks post infection along with marked improvement indisease pathology as evidenced by reduced organ weight and pathologyscores. However, the extent of reduction in bacillary burden in case ofrBCG-disAOE immunization was markedly greater (by 0.37 log₁₀ in lung and1.6 logic in spleen), when compared to BCG immunized animals. Further,at 18 weeks post-infection along with a markedly reduced diseasepathology, rBCG immunized animals exhibited a markedly reduced lung (by2.49 log₁₀) and spleen (by 4.68 log₁₀) bacillary burden when compared tosham immunized animals. Most importantly, rBCG treated animals showed amarked improvement when compared to BCG treated animals both in terms oflung and spleen bacillary burden (by 1.9 log₁₀ and 2.54 logic,respectively). [See attached Figures]

EXAMPLES

Bacterial Strains, Plasmids, Cell Lines and Animals.

In this study we used Escherichia coli strain DH5α, M. tuberculosisCDC1551, M. tuberculosis Erdman, M. bovis bacillus Calmette-Guérin(BCG). FIG. 14 lists all the Mycobacterium strains and plasmids used inthis study. Details on the transposon insertion mutant of M.tuberculosis for MT3692 (M. tuberculosis-mutant JHU-3586; Rv3586) usedin this study is available on the TARGET website of the Johns HopkinsUniversity. Plasmid pSD5-hsp60 (mycobacteria—E. coli shuttle vector forprotein expression in M. tuberculosis from the strong mycobacterialpromoter, hsp60) was used for expression. Plasmid pMH94Hyg was used forcomplementation. Both E. coli and mycobacterial strains were grown fromfrozen glycerol stocks stored at −70° C. Murine macrophage cells J774.1,RAW 264.7-derived macrophages such as RAW-Blue ISG, RAW-Lucia ISG,RAW-Lucia ISG-KO-STING (Sting knockout cells), human monocyte THP1-Dual,THP1-Blu ISG-KO-STING cells (all from InvivoGen) and primary BMDMs andBMDCs from C57BL/6J and cGAS-KO mouse were used for in vitroexperiments. All cell lines are free of mycoplasma contamination. Femalemouse strains BALB/c and CS7BL/6J (Jackson laboratories), age 6-7 weeks,were used for comparative studies of bacterial virulence, pathogenicityand time to death. The experiments were approved by the InstitutionalAnimal Care and Use Committees (IACUCs) of Johns Hopkins University.

Reagents.

1774.1 cells were cultured in RPMI-GlutaMAX (Life Technologies) with 10%(vol/vol) heat-inactivated FBS (Life technologies). Variants of RAW264.7and THP-1 cells were cultured as per the suppliers protocol (InvivoGen)in DMEM and RPMI-GlutaMAX, respectively (Life Technologies) with 10%heat-inactivated FBS. The following antibodies were used: antibody toDDX41 (anti-DDX41; G14; Santa Cruz Biotechnology Inc. 1:500);anti-LC3A/B (D3U4C, XP; Cell Signaling Technologies, 1:1,000);anti-STING (D2P2F; Cell Signaling Technologies, 1:1,000); anti-pTBK1(Seri 72, D52C2; Cell Signaling Technologies 1:1,000); anti-GAPDH(14C10; Cell Signaling Technologies, 1:2,000). Secondary antibodyanti-rabbit conjugated to fluorochrome Alexa Fluor 488, DAPI (LifeTechnologies), c-di-AMP (BIOLOG Life Science Institute), c-di-UMP,puromycin, zeocin, blasticidin, Quanti-Blue, Quanti-Luc (InvivoGen),hygromycin (Roche) and kanamycin (Sigma-Aldrich).

Extraction of Nucleotides from M. tuberculosis and Detection andQuantitation of c-Di-AMP by LC-MS/MS/MRM.

Nucleotide extraction for LC-MS/MS were carried out as described 42.Briefly, M. tuberculosis was grown to mid-log phase and harvested byquick centrifugation followed by resuspension in extraction buffercontaining acetonitrile and methanol and water (2:2:1) and cXMP as aninternal technical control. After a 15-min incubation and subsequentboiling at 100° C. for 10 min, the mixture was cooled down and extractedafter quick centrifugation. Extraction was repeated twice as describedabove. Pooled samples were vacuum dried and resuspended in distilledwater followed by detection and quantitation by LC-MS/MS MRM. Briefly,the chromatographic separation was performed on a Series 200 HPLC system(Perkin Elmer Instruments) and the analyte detection was performed on anAPI 3000 triple quadrupole mass spectrometer equipped with anelectrospray ionization (ESI) source (Applied Biosystems Inc.) using MRManalysis in positive ionization mode. The following SRM transitionsusing a dwell time of 40 ms were detected: cXMP: +347.1/153(quantifier), +341.7/136 (identifier) and c-di-AMP: +659.1/330.2(quantifier) and +659/524 (identifier).

Extraction of Nucleotides from Macrophage Cytoplasm and Detection andQuantitation of c-di-AMP by LC-MS/MS/MRM.

J774.1 cells were cultured in RPMI medium with 10% heat-inactivated FBS.Infections were carried out in either resting or IFN-γ- andLPS-activated J774.1 cells in six-well plates in triplicate. Forinfection, early log-phase cultures of various Mycobacteriumtuberculosis strains were washed and diluted appropriately to predefinedconcentrations in antibiotic-free RPMI and were added to the J774.1cells at a precalibrated MO. The infection was allowed to continue for 4h, following which extracellular bacteria were removed by washing theinfected cells with DPBS thoroughly. After 24 h of infection,supernatants were removed and adherent macrophages were washed carefullywith DPBS. Macrophages were lysed with the addition of 1 ml of 0.025%SDS (at this concentration of SDS, bacteria is not lysed) to each of thewells. Released bacilli were subsequently separated by centrifugationfollowed by filtration through a 0.2-μm membrane filter, andbacteria-free pooled macrophage cytoplasmic extracts were used forextraction of nucleotides and subsequent analysis by LC-MS/MS asdescribed above.

Overexpression of MT3692 in M. tuberculosis.

The disA4 gene of M. tuberculosis, MT3692, was PCR-amplified from M.tuberculosis CDC1551 chromosomal DNA using gene-specific primers,pSD5hsp60.MT3692(F) and pSD5hsp60.MT3692(R). The amplicons were clonedinto the Mycobacterial expression vector pSD5-hsp60 at the NdeI and MluIrestriction sites. The resulting construct pSD5-hsp60-MT3692 wassequenced and subsequently used to transform M. tuberculosis CDC1551 andrecombinant clones were selected against kanamycin and confirmed bycolony PCR using kanamycin gene-specific primers. Overexpression ofMT3692 in the Mtb-OE strain was further confirmed by RNA sequencing ofthe Mtb-OE strain and measurement of c-di-AMP by LC-MS-MRM.Overexpression of MT3692 in the M. tuberculosis Erdman and M. bovis BCGstrains were carried out using the same plasmid.

Construction of MT3692 Complementation Strain.

To complement the transposon mutant for MT3692, a 279-bp DNA fragmentincluding the coding sequence of the MT3692 gene and 1,714 bp of the 5′sequence (including the upstream gene in the operon and gene's nativepromoter) was amplified by PCR with primers OPE-MT3692(F) andOPE-MT3692(R) and cloned into an integretion vector, pMH94Hyg, at anXbaI restriction site. The resulting construct, pMH94Hyg-MT3692, wassubjected to nucleotide sequencing and subsequently used to transformthe Mtb-disA-KO strain. Candidate Hygromycin resistant Mtb-COMP colonieswere selected, confirmed by PCR using hygromycin gene-specific primersand geniomic DNA as template. Mtb-COMP clones were further confirmed bymeasurement of c-di-AMP by LC-MS/MRM method.

Infection of Mice with M. tuberculosis and Assessment of Bacterial Load,Pathology and Time to Death.

Four strains of M. tuberculosis, Mtb-disA-KO, Mtb-COMP, Mtb-OE andMtb-WT were used to infect 6-7-week-old female C57BL/6J mice by theaerosol route in a Glascol inhalation exposure system (Glascol) with aninoculum that implanted ˜3.0 Log 10 c.f.u. in the lungs at day 1 (n=3mice in each group). Animals from a narrow range of weight and agegroups were randomly allocated for infection with different bacterialstrains. Eight mice from each group were subsequently sacrificed at 2,4, 8 and 12 weeks after infection to determine the lung and spleenc.f.u. counts (n=4) and histopathology and immunology studies (n=4).Lung and spleen tissues were homogenized in their entirety in PBS andcolonies were enumerated on selective 7H11 plates after 3-4 weeks ofincubation at 37° C. The number of colonies were counted and expressedas log 10 c.f.u. per organ. All groups were coded during theexperiments. For histopathology, whole lungs were fixed in 10% bufferedformalin and sections of 5 μm in thickness from formalin fixed andparaffin embedded tissues were cut onto glass slides and stained withH&E for histopathological examination. For time to death assay6-7-week-old female BALB/c mice (n=10 per group) were infected asdescribed above with ˜3.5 log 10 c.f.u. of various strains of M.tuberculosis and monitored until their death due to tuberculosis. Allexperiments were carried out according to the guidelines of theInstitutional Animal Care and Use Committees (IACUCs) of Johns HopkinsUniversity.

Infection of Macrophages with M. tuberculosis and Assay for IRFActivation and IFN-b Production.

J774.1 cells were cultured in RPMI medium with 10% heat-inactivated FBS.Infections were carried out in either resting or IFN-γ- andLPS-activated J774.1 cells in 24-well plates in triplicate. Forinfection, early log-phase cultures of various M. tuberculosis strainswere washed and diluted appropriately to predefined concentrations inantibiotic-free RPMI and were added to the J774.1 cells at aprecalibrated MOI. The infection was allowed to continue for 4 h,following which extracellular bacteria were removed by washing theinfected cells with DPBS thoroughly. Serial dilutions of the bacterialsuspension and macrophage lysate were plated at day ‘0’ in order todetermine an accurate bacterial count of infection and phagocytizedbacterial number. For enumeration of bacterial growth, at 1, 2 and 4 dafter infection cells were harvested and lysed using 0.025% SDS.Appropriate dilutions of the lysates were then inoculated onto MB7H11agar plates in duplicate and incubated at 37° C. for 3 weeks. The numberof colonies was counted and expressed as log 10 c.f.u, per well.Investigators were blinded for c.f.u. analysis. Macrophage culturesupernatants collected at the indicated time points were used formeasurement of various cytokines by ELISA. For immunofluorescence andwestern blot detection of LC3, at 6 h after infection macrophage cellswere washed thoroughly and either fixed in 4% paraformaldehyde in PBSfollowed by immunofluorescence staining or lysates were prepared in RIPAbuffer (Cell Signaling Technologies) for western blotting. RAW-Blue ISGand RAW-Lucia ISG or RAW-Lucia ISG-KO-STING (InvivoGen) cells werederived from the murine RAW 264.7 macrophage cell line by stableintegration of an Interferon regulatory factor (RF)-inducible secretedembryonic alkaline phosphatase (SEAP) and luciferase reporterconstructs, respectively. These cells without prior activation wereinfected with various strains of M. tuberculosis with a pre-calibratedMOI of 1:5 for 4 h. After infection, extracellular bacteria were removedby washing the infected cells with DPBS thoroughly. After 18 hincubation in fresh DMEM, supernatants were collected for estimation ofIRF induction by SEAP colorimetric assay using QUANTI-Blue reagent(InvivoGen) or Luminescence assay using QUANTI-Luc (InvivoGen) and formeasurement of cytokines by ELISA. THP1-Dual cells (InvivoGen) weregrown as per the suppliers recommendations. THP1-Dual cells were derivedfrom the human THP-1 monocyte cell line by stable integration of twoinducible reporter constructs, a new secreted luciferase reporter gene,under the control of an ISG54 (interferon-stimulated gene) minimalpromoter in conjunction with five IFN-stimulated response elements and aSEAP reporter gene fused to five copies of the NF-kB consensustranscriptional response element and three copies of the c-Rel bindingsite. As a result, THP1-Dual cells allow the simultaneous study of theNF-kB pathway, by monitoring the activity of SEAP, and the IRF pathway,by assessing the activity of Lucia in culture supernatants. THP1-BlueISG-KO-STING cells were generated from THP1-Blue ISG cells throughknockdown of the STING gene, and they were cultured as per thesupplier's recommendations (InvivoGen). Mouse primary BMDMs and BMDCswere cultured and infected with precalibrated MOIs as described abovefor immortalized cell lines.

shRNA-Mediated Interference.

RAW Blue ISG (InvivoGen) cells were transfected with a pool of fivelentiviral vectors carrying a target gene sequence for DDX41 or acontrol plasmid (pLKO.1) (Thermo Scientific). At 24 h aftertransfection, cells were selected by the addition of puromycin to themedium. For transfection Lipofectamine LTA Plus (Life Technologies)reagent was used as per the manufacturer's instructions. Knockdown ofDDX41 was confirmed by western blotting.

ELISA.

ELISAs for IFN-β, IFN-γ, IL-1α, IL-6 and TNF-α were performed with themacrophage cell culture supernatants and serum of infected mice by usingmouse cytokine-specific ELISA kits (eBiosciences, Biolegend) as permanufacturers' instructions. In vitro macrophage culture experimentswere carried out in triplicate and at least thrice. Serum from four micein each group were assayed by ELISA for cytokine levels.

Western Blot Analysis.

For immunoblot analysis, macrophage cells at predefined time pointsafter infection were collected and lysed in RIPA lysis buffer (CellSignaling Technologies) containing complete protease inhibitors (Roche).LC3, STING, DDX41 and GAPDH western immunoreactivity assays ofmacrophage lysates were performed using anti-mouse antibodies per theantibody provider's (Cell Signaling Technology). Densitometry analysesof the western blots were carried out with GelQuant software.

Two-Color Immunofluorescence and Confocal Microscopy.

Immunofluorescence staining was carried out by serial incubation offixed cells grown on culture slide chambers with LC3-specific antibody,(Cell Signaling Technology) followed by incubation with anisotype-specific, fluorochrome (Alexa Fluor 488)-labeled goatanti-rabbit antibody (A-11001; Molecular Probes). Nuclei were stainedwith DAPI. For imaging, we used an Olympus BX61 with Roper/PhotometricsCoolsnap HQ fluorescence microscope and Zeiss LSM 510-mets confocallaser-scanning microscope at the Johns Hopkins University coremicroscopy facility. Slidebook (Intelligent Imaging), ZenLite (Zeiss)and ImageJ (public domain software available from the US NationalInstitutes of Health) software were used for image acquisition and/oranalysis. Investigators were blinded during analysis. For LC3 analysis astringent threshold was set to define a ‘puncta’, such that only thosecells that exhibited formation of large LC3 aggregates occupying anarea >1 μm were considered as positive. Extent of autophagy induction isthus represented by the percentage of LC3-positive cells.

Real-Time RT-qPCR.

Twenty four hours after infection with different strains of M.tuberculosis, RNA was extracted using the RNeasy Plus Micro kitaccording to the manufacturer's protocol (Qiagen). RNA wasreverse-transcribed using the iSoript Reverse Transcription Supermix(Bio-Rad) containing oligo-dT and random primers. cDNA was used forreal-time qPCR using 2×iQ SYBR Green Supermix and an iCycler (Bio-Rad).The primers for real-time RT-qPCR. The IFN-β mRNA expression levels werenormalized to β-actin expression and fold induction was calculated bythe ΔΔCT method relative to those of untreated cells.

Statistical Analyses.

For comparisons between groups, Student's t-test (two tailed), one-wayANOVA with Tukey's post-test and two-way ANOVA with Bonferroni post-testwere used wherever appropriate. Differences were considered significantat at least P<0.05. For statistical analysis, we used Prism 5 software(Version 5.01; GraphPad Software Inc.).

The invention claimed is:
 1. A pharmaceutical composition comprising astrain of Mycobacterium selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovisbacillus Calmette Guerin BCG, and a combination thereof comprising anexpression vector encoding a di-adenylate cyclase enzyme and apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1 wherein the strain of Mycobacterium is BCG.
 3. Thepharmaceutical composition of claim 1 wherein the expression vector is amycobacterial expression vector.
 4. The pharmaceutical composition ofclaim 3 wherein the expression vector comprises the DNA sequence ofdiadenylate cyclase (disA).
 5. The pharmaceutical composition of claim4, wherein the disA is that of M. tuberculosis (Rv3586).
 6. Thepharmaceutical composition of claim 1 wherein the expression vectorcomprises a hsp60 promoter.
 7. The pharmaceutical composition of claim 6wherein the expression of di-adenylate cyclase enzyme is regulated bythe hsp60 promoter.
 8. The pharmaceutical composition of claim 1 furthercomprising at least one or more compounds enhancing immunogenicity. 9.The pharmaceutical composition of claim 8 wherein the at least one ormore compounds enhancing immunogenicity is a synthetic c-di-AMP.
 10. Thepharmaceutical composition of claim 8 wherein the at least one or morecompounds enhancing immunogenicity is selected from the group consistingof mycobacterial DNA, IFN, and combinations thereof.